Entomopoxviruses (EPVs) are insect-specific members of the family Poxyiridae (Murphy et al., 1995) that collectively infect hosts such as caterpillars, beetles and locusts (Arif, 1995). Like other members of the poxvirus family (i.e., the chordopoxviruses; ChPVs), EPVs have large double-stranded DNA genomes, produce complex virions, and replicate in the cytoplasm of infected cells (Moss, 1996). While these and other molecular characteristics confirm their poxvirus affinities (Osborne et al., 1996), other notable traits differentiate EPVs from ChPVs, and ally them instead with unrelated groups of insect-infecting viruses. Foremost among these traits is production of the distinctive proteinaceous structures known as spheroids and spindle bodies.
Spheroids develop in the cytoplasm of EPV-infected cells at the site of viral morphogenesis, and when mature, occlude large numbers of infectious virions (Goodwin et al., 1991). They are the agent of horizontal transmission of EPVs, and while their major constituent matrix protein (spheroidin; Hall & Moyer, 1991) has no known homologue outside the taxon, the bodies themselves are assumed to protect virions from detrimental environmental factors such as desiccation and exposure to u.v. light. In this respect they are functionally analogous to the polyhedral bodies which occlude virions of members of the baculovirus family and the cytoplasmic polyhedrosis group of reoviruses.
Most EPVs also encode and produce a protein known as fusolin, which has been shown to be the major constituent of structures known as spindle bodies (SBs; Dall et al., 1993); these structures have been described from many, but not all, members of EPV genera A and B that infect caterpillars and beetle larvae (Goodwin et al., 1991). In the Heliothis armigera EPV (HaEPV)(Fernon et al., 1995), the fusolin protein has a calculated Mr of 40132, and the mature form of the protein has an apparent size of 50K when analysed by SDS-PAGE (Dall et al., 1993). The protein has been found to accumulate in vesicular structures derived from cellular endoplasmic reticulum, where it eventually aggregates and crystallises into SBs (Lai-Fook and Dall, in press). Although other proteins are known to be co-located in SBs (e.g., the ER-specific chaperone protein, BiP; Lai-Fook and Dall, in press), analysis of purified SB preparations shows that fusolin, in its monomeric and multimeric forms (Dall et al., 1993), is by far the most abundant constituent.
Genes encoding homologues of the fusolin protein, in this context known variously as “gp37”, “37K protein”, “SLP” (spindle-like protein), etc., have also been described from a number of nuclear polyhedrosis (NPV) baculoviruses, including the Autographa californica, Bombyx mori, Choristoneura fumiferana, Lymantria dispar, Orgyia pseudotsugata NPVs and Xestia c-nigrum GV (AcMNPV, BmMNPV, CfMNPV, LdMNPV, OpMNPV and XcGV, respectively; Ayres et al., 1994; Gomi et al., 1999; Liu and Carstens, 1996; Kuzio et al., 1999; Ahrens et al., 1997; Hayakawa et al., 1999). In some of these (e.g., OpNPV; Gross et al., 1993), the protein has been observed within spindle-like bodies (SLBs) in the cytoplasm of infected cells. SLBs have also been observed in the cytoplasm of cells infected with other NPVs (e.g., from Cadra cautella NPV, Adams and Wilcox 1968; see also Adams and McClintock, 1991; Cunningham, 1971; Huger and Kreig, 1968; Smirnoff, 1970).
All members of the fusolin group of proteins, irrespective of their viral family of origin, are united by an absolute conservation of amino acid residues at a number of positions in their sequences, in particular in the N-terminal and central regions of the molecule. These conserved residues include HGX (standard one letter amino acid code, where X is an aromatic amino acid) and ARQ motifs near the N-terminal of the deduced protein sequence (Table 1), and e.g. a VRWQR (SEQ ID NO:1) sequence elsewhere within the deduced amino acid sequence (FIG. 1). This conservation of sequence elements, like that of the protein's intracellular location, as previously described, suggests that all members of the group also share a common role in the cycle of virus infection and replication, perhaps in influencing the relationship of the viruses with their hosts (Sriskantha et al., 1997). Nevertheless, the function(s) of members of this group of proteins, and the SB/SLB structures that they form, remain a topic of on-going investigation.
Studies by Xu and Hukuhara (1992, 1994) suggested that a factor associated with preparations of Pseudaletia separata EPV (PsEPV), and subsequently identified as fusolin (Hayakawa et al., 1996), was capable of enhancing the infectivity of a heterologous nuclear polyhedrosis virus (P. unipunctata NPV). Further studies have shown that a similar effect can be seen in transgenic rice plants in which this protein has been expressed (Hukuhara et al., 1999). Similarly, the SBs of the cupreous chafer (Anomala cuprea) have been shown to be capable of acting in the same manner (Mitsuhashi et al., 1998). The role(s) of fusolin protein in the context of homologous EPV systems has not, however, been previously subjected to detailed investigation.
Through experiments involving bioassays using SBs of Heliothis armigera EPV (HaEPV) and Dermolepida albohirtum EPV (DaEPVSR), the present applicants have determined, unexpectedly, that consumption of spindle bodies alone can effect feeding, growth and development of insect larvae. Further, through experiments conducted using recombinant EPVs wherein the fusolin gene has been replaced with a β-galactosidase marker (i.e., to render the recombinant EPVs fusolin negative [fus(−)]), the present applicants have also been able to provide evidence to show that it is the fusolin protein component of SBs that is responsible for these effects. Moreover, the latter experiments have indicated that fusolin enhances the infectivity of the homologous EPV virus. As a result, it has been realised that SBs, SLBs and constituent proteins of these structures may be advantageously used in strategies designed to reduce damage caused to plants by feeding insects.